The dendritic cell (DC) C-type lectin DC-SIGN can function in both cell adhesion and antigen recognition, and several pathogens appear to interact with DC-SIGN for their own benefit. DC-SIGN mediates myriad responses depending on the ligand it binds, and the molecular mechanisms that underlie different cellular responses are not understood. As some pathogens bind DC-SIGN to promote infection, inhibitors of DC-SIGN may function as novel antimicrobial agents. In addition, a better understanding of the mechanisms by which DC-SIGN mediates antigen uptake and presentation to T cells could lead to enhanced vaccine strategies. DC- SIGN is a carbohydrate-binding protein with relatively weak affinity for a set of mannose- and fucose- based glycans, and DCs also express other receptors that bind similar structures. Thus, it has been difficult to dissect the role of any one lectin. The goal of the proposed research is to elucidate the role of DC-SIGN in health and disease by describing the fate of ligands with different properties that bind DC-SIGN. Ligands that display high affinity for DC-SIGN have been generated, and these will be employed. These ligands can be labeled with a fluorogenic reporter to allow their cellular uptake and trafficking to be observed in real time in live cells. Such ligands will be used to accomplish the following Specific Aims: 1. Compare ligand specificity of DC-SIGN to that of other lectins. Ligands found to bind with high affinity to DC-SIGN will be tested for binding to additional C-type lectins present on dendritic cells. These additional lectins will be screened against a library of glycomimetic compounds to assess inhibitor specificity among the different receptors. These studies will also yield high-affinity inhibitors for further study of several immune cell lectins, in addition to DC-SIGN. 2. Determine the effect of ligand structure on DC-SIGN function. Ligands of varying properties, such as low or high valency, will be used to treat cells to study the downstream outcomes of binding to DC-SIGN. Outcomes of interest include the rate of antigen uptake and intracellular routing. These investigations will be carried out using transfected cells that lack other immune C-type lectins, to ascertain the function of DC- SIGN in isolation. 3. Explore the consequences of DC-SIGN co-activation with other receptors. The possibility that multiple receptors cooperate to control responses will be evaluated by using compounds that can engage multiple receptors simultaneously. The cellular uptake and intracellular routing will be evaluated using immune cells that also express additional receptors. This work will greatly increase our understanding of how DC-SIGN functions in immunity. I anticipate that it also will provide new probes for studying DC-SIGN and perhaps even other C-type lectins in the immune system. PUBLIC HEALTH RELEVANCE: Project Narrative DC-SIGN is a receptor on the surface of dendritic cells that can both recognize pathogens and promote interactions with other immune cells. We seek to elucidate the function of this receptor in terms of interactions with pathogens. A better understanding of how these types of receptors function may allow us to manipulate them for the purpose of killing pathogens, making better vaccines, or targeting immune cells to clear tumors.